Enamel matrix proteins, present in the enamel matrix, are most well-known as precursors to enamel. Prior to cementum formation, enamel matrix proteins are deposited on the root surface at the apical end of the developing tooth-root. There is evidence that the deposited enamel matrix is the initiating factor for the formation of cementum. Again, the formation of cementum in itself is associated with the development of the periodontal ligament and the alveolar bone. Enamel matrix proteins can therefore promote periodontal regeneration through mimicking the natural attachment development in the tooth (Gestrelius S, Lyngstadaas S P, Hammarström L. Emdogain—periodontal regeneration based on biomimicry. Clin Oral Invest 4:120-125 (2000)).
Isolated enamel matrix proteins are able to induce not only one, but an orchestrated cascade of factors, naturally found in tissues developing adjacent to the enamel matrix. They mimic the natural environment of a developing tissue and thus mimic a natural stimulation for tissue regeneration, cell differentiation and/or maturation.
Enamel matrix derivative (EMD), in the form of a purified acid extract of proteins from pig enamel matrix, has previously been successfully employed to restore functional periodontal ligament, cementum and alveolar bone in patients with severe tooth attachment loss (Hammarström et al., 1997, Journal of Clinical Periodontology 24, 658-668).
Enamel matrix derivative (EMD) formulations have also been shown to promote periodontal regeneration (Hammarström et al., 1997, Journal of Clinical Periodontology 24, 669-677). In this study different formulations were used in order to find out if the result would differ depending on which formulation was used. The study showed that the most satisfactory result was given when EMD was dissolved in PGA.
For example, U.S. Pat. No. 5,098,891 describes for the first time a composition for use in inducing binding between parts of mineralized tissue by regeneration of mineralized tissue on at least one of the parts, containing as an active constituent a protein fraction originating from a precursor to dental enamel, so called enamel matrix.
Furthermore, in studies on cultured periodontal ligament cells (PDL), it was shown that the attachment rate, growth and metabolism of these cells were significantly increased when EMD was present in the cultures. Also, cells exposed to EMD showed increased intracellular cAMP signaling and autocrine production of growth factors when compared to controls. Epithelial cells, on the other hand, although increasing cAMP signaling and growth factor secretion when EMD was present, were inhibited in both proliferation and growth (Lyngstadaas et al., 2001, Journal of Clinical Periodontology 28, 181-188).
Enamel matrix proteins and enamel matrix derivatives (EMD) proteins have previously been described in the patent literature to be able to induce hard tissue formation (i.e. enamel formation, U.S. Pat. No. 4,672,032 (Slavkin)), endorse binding between hard tissues (EP-B-0 337 967 and EP-B-0 263 086), promote open wound healing, such as of skin and mucosa, have a beneficial effect on treatment of infections and inflammatory diseases (EP-B-1059934 and EP-B-1153610), induce regeneration of dentin (WO 01/97834), promote the take of a graft (WO 00/53197), induce apoptosis in the treatment of neoplasms (WO 00/53196), regulate imbalance in an immune response to a systemic infection or inflammation (WO 03/024479), and to facilitate filling a wound cavity and/or tissue defect following from a procedure and/or trauma, such as a cytoreductive surgery (WO 02/080994).
EMD is composed of a number of proteins, such as amelogenins, enamelin, tuft protein, proteases, and albumin. Amelogenins, a major constituent of EMD, at least up to 60%-90%, such as 70-90%, are a family of hydrophobic proteins derivable from a single gene by alternative splicing and controlled post secretory processing. They are highly conserved throughout vertebrate evolution and demonstrate a high overall level of sequence homology among all higher vertebrates examined (80%). In fact, the sequences of porcine and human amelogenin gene transcript differ only in 4% of the bases. Thus, enamel matrix proteins and/or EMD proteins, although of porcine origin, are considered “self” when encountered in the human body and can promote dental regeneration in humans without triggering allergic responses or other undesirable reactions. Enamel Matrix Derivative Protein (EMD) is the most known precursor to enamel. Its aqueous solution thickened with PGA is commercialized under the trade-name Straumann® Emdogain. Enamel Matrix Derivative Protein (EMD) aqueous solution thickened with PGA can also be found in Straumann® Emdogain Plus.
In order to keep the EMD in aqueous solution, the solution should have a pH well-below the protein isoelectric point (IEP), for EMD the IEP is pH 6.5, hence, more preferred is that the pH of the solution is <5.0. For easy application, the solution is thickened by PGA. Chemically, the PGA is an ester of alginic acid, which is derived from kelp (seaweed). Some of the carboxyl groups are esterified with propylene glycol, some are neutralized with an appropriate alkali, and some remain free. The PGA itself is acidic and the pH decreases after the PGA is dissolved in the EMD solution. Under acidic conditions, the pH keeps decreasing due to the degradation of PGA. The durability of the mixture is determined by the pH value at which the EMD is capable to precipitate onto the tooth surface. It is considered that the precipitation occurs in physiological conditions, i.e. pH near IEP. The pH and buffering capacity of both the tooth root environment and the EMD-PGA mixture influences the precipitation behavior of the EMD proteins, a pH near IEP and a lower buffering capacity of the EMD solution formulated with PGA favors the precipitation.
D. J. McHugh describes in HYPERLINK http://www.fao.org/docrep/x5822e/x5822e04.htm, that the degree of polymerization (DP) of an alginate is a measure of the average molecular weight of the molecules and is the number of uronic acid units per average chain. DP and molecular weight relate directly to the viscosity of alginate solutions; loss of viscosity on storage is a measure of the extent of de-polymerization of the alginate.
PGA is produced in various grades, which are usually described as low, medium and high viscosity alginates (referring to the viscosity in 2% aqueous solution). The higher the molecular weight of a PGA alginate, the greater the viscosity of its solution. Manufacturers can control the molecular weight (degree of polymerization, DP) by varying the severity of the extraction conditions and they offer products ranging from 10-1000 mPa·s (1% solution) with a DP range of 100-1 000 units. PGA of viscosity 200-400 mPa·s, “medium viscosity”, probably finds the widest applications. PGAs with a high DP are known to be less stable than those with a low DP. Low viscosity PGA (up to about 50 mPa·s) has been stored at 10-20° C. with no observable change in 3 years. Medium viscosity sodium alginates (up to about 400 mPa·s) show a 10% loss at 25° C. and 45% loss at 33° C. after one year, and higher viscosity alginates are even less stable.
Propylene glycol alginates showed about 40% loss in viscosity after a year at 25° C. and also became less soluble. Ammonium alginate is generally less stable than any of the above. Alginic acid is the least stable of the products and any long chain material degrades to shorter chains within a few months at ambient temperatures. However, alginates comprising short chain material are stable and alginic acid with a DP of about 40 units of uronic acid per chain will show very little change over a year at a temperature of 20° C. However, the main use of alginic acid, as a disintegrant in pharmaceutical tablets, depends on its ability to swell when wetted and this is not affected by changes in DP. The commercial alginates should be stored in a cool place, i.e. at temperatures of 25° C. or lower as elevated temperatures can cause significant depolymerization, affecting the commercially useful properties, such as viscosity and gel strength. Said alginates usually contain 10-13% moisture and the rate of depolymerization increases as the proportion of moisture is increased, thus the storage area should be dry.
As mentioned above, EMD proteins have prior been formulated in an aqueous solution with PGA, wherein the degradation of PGA provides acidic products, which in turn decreases the pH over time. At elevated temperatures, the degradation accelerates. In order to avoid the accelerated acidification of the product, and thus to limit this effect, the products have to be transported and stored at low temperatures, i.e. at a temperature range of 2 to 8° C. Nonetheless, the stability of EMD in the commercially available Straumann® Emdogain formulation with PGA has been known to decrease rapidly over time.